We, along with others, have shown increased levels of fucosylation with the development of hepatocellular carcinoma (HCC). In an effort to determine the origin of this increased fucosylation, we have performed N-linked glycan analysis of HCC tissue, the surrounding non tumor tissue, and compared this to tissue from a non-diseased adult liver. Surprisingly, no difference in the level of fucosylation was observed from the three donor groups, suggesting that the increased levels of fucosylation observed in the serum of those with HCC is not the result of increased synthesis of fucosylated proteins in the cancer tissue. In addition, the level of fucosylation observed in tissue was much higher than that observed on liver derived serum glycoproteins. On the other hand, increased levels of a tetra-antennary glycan were observed in the HCC tissue as compared to the surrounding tissue or to the non-diseased livers. Recent work by our collaborator has suggested that fucosylation controls the polarized secretion of glycoproteins and directs their secretion apically into the bil. Indeed, bile glycoforms are more heavily fucosylated than in serum and closely resemble those observed in the serum of HCC patients. It is our hypothesis that the over-expression of 1,6-N-acetylglucosaminyltransferase V (MGAT-5), which is responsible for the increased branching we have observed, leads to a loss of a tight blood-biliary barrier and the release of fucosylated proteins into the blood. This hypothesis, along with the use of this knowledge to find new biomarkers of HCC, will be tested in 3 aims. First we will examine the role of MGAT-5 in enforcing the polarized secretion of core fucosylated proteins. It is hypothesized that the overexpression of MGAT-5 that we observe in HCC tissue leads to a defect in the blood- biliary barrier and the aberrant release of fucosylated proteins into the blood. In specific aim 2 we will perform glycoproteomic analysis of bile for markers of HCC. The logic here is that all hepatocytes have the ability to secrete into the blood stream or into the bile capillaries, which eventually forms the bile ducts. Loss of hepatocyte polarity will lead to the aberrant appearance of fucosylated proteins in the blood. In specific Aim 3 we will examine the ability of our identifid biomarkers to differentiate patients with HCC from patients with liver cirrhosis in a cohort of 1500 patients. The ability of these markers to distinguish HCC from cirrhosis as compared to AFP and other potential biomarkers of HCC will be determined. PUBLIC HEALTH RELEVANCE: The overall goal of this project is an understanding of how changes in glycosylation promote cancer progression and to use this information to identify glycan-based biomarkers for the early detection of cancer.